2.1 Technical Field
The present invention provides novel polynucleotides and proteins encoded by such polynucleotides, along with uses for these polynucleotides and proteins, for example in therapeutic, diagnostic and research methods. In particular, the invention relates to novel alpha-2-macroglobulin-like polypeptides.
2.2 Background Art
Identified polynucleotide and polypeptide sequences have numerous applications in, for example, diagnostics, forensics, gene mapping; identification of mutations responsible for genetic disorders or other traits, to assess biodiversity, and to produce many other types of data and products dependent on DNA and amino acid sequences. Proteins are known to have biological activity, for example, by virtue of their secreted nature in the case of leader sequence cloning, by virtue of their cell or tissue source in the case of PCR-based techniques, or by virtue of structural similarity to other genes of known biological activity. It is to these polypeptides and the polynucleotides encoding them that the present invention is directed. In particular, this invention is directed to novel alpha-2-macroglobulin-like polypeptides and polynucleotides.
Alpha-2-macroglobulin is a highly conserved proteinase inhibitor present in plasma at relatively high concentrations (2-4 mg/ml). It is unique in its ability to inhibit all the major classes of proteinases (Bhattacharjee et al (2000) J. Biol. Chem. 275, 26806-26811). Alpha-2-macroglobulin is a tetramer of four identical 180 kDa subunits that forms a hollow cylinder-like structure. It presents multiple target peptide bonds to attacking proteinases in its central xe2x80x9cbaitxe2x80x9d domain. Binding of the proteinase and subsequent cleavage of the bait domain leads to a conformational change trapping the proteinase in the central cavity. The xe2x80x9cactivatedxe2x80x9d alpha-2-macroglobulin is now recognizable by its receptor. The receptor bound activated alpha-2-macroglobulin is then endocytosed, thus removing the potentially harmful proteinases from the circulation. Alpha-2-macroglobulin is the major proteinase inhibitor acting on foreign proteinases like snake venoms.
However, there are many other proteinase inhibitors in the circulation and it is proposed that alpha-2-macroglobulin may have other functions including binding to and regulation of cytokine and growth factor activity, promotion of tumoricidal capabilities of macrophages, and enhancement of antigen presentation. In accordance with above findings, numerous cytokines, growth factors and other plasma proteins have been shown to bind to alpha-2-macroglobulin including transforming growth factor-beta, platelet-derived growth factor, nerve growth factor, tumor necrosis factor-alpha, basic fibroblast growth factor, interleukin-6, beta-amyloid peptide, apolipoprotein E, myelin basic protein, leptin, beta-microglobulin and vascular endothelial growth factor (VEGF). Most of these proteins do not bind to the bait domain. Also, the native and activated alpha-2-macroglobulins show different binding specificities and kinetics. Binding of these proteins to alpha-2-macroglobulin could sequester them from proteinases and help maintain effective concentrations.
Epidemiological and genetic studies have indicated that alpha-2-macroglobulin deficiency and/or polymorphism may be associated with Alzheimer""s disease and cerebral amyloid angiopathy, multiple sclerosis, and congenital antithrombin deficiency. Alpha-2-macroglobulin and VEGF are also implicated in rheumatoid arthritis and growth of various tumors, coronary or limb ischemia and retinopathies. Binding of alpha-2-macroglobulin to beta-microglobulin could be involved in regulation of immune response to tumors and viral infections. It is conceivable that alpha-2-macroglobulin exerts its effects by binding to various biologically important molecules and regulating their activities.
Thus, the alpha-2-macroglobulin-like polypeptides and polynucleotides of the invention may be used in the treatment of Alzheimer""s disease and cerebral amyloid angiopathy, multiple sclerosis, congenital antithrombin deficiency, rheumatoid arthritis and growth of various tumors, coronary or limb ischemia, retinopathies, and regulation of immune response to tumors and viral infections.
This invention is based on the discovery of novel alpha-2-macroglobulin-like polypeptides, novel isolated polynucleotides encoding such polypeptides, including recombinant DNA molecules, cloned genes or degenerate variants thereof, especially naturally occurring variants such as allelic variants, antisense polynucleotide molecules, and antibodies that specifically recognize one or more epitopes present on such polypeptides, as well as hybridomas producing such antibodies. Specifically, the polynucleotides of the present invention are based on an alpha-2-macroglobulin-like polynucleotide isolated from a cDNA library prepared from fetal brain (Clontech) (SEQ ID NO: 1) and from uterus (Clontech) (SEQ ID NO: 26).
The compositions of the present invention additionally include vectors such as expression vectors containing the polynucleotides of the invention, cells genetically engineered to contain such polynucleotides and cells genetically engineered to express such polynucleotides.
The compositions of the invention provide isolated polynucleotides that include, but are not limited to, a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 1-3, 5, 26-28, or 30; or a fragment of SEQ ID NO: 1-3, 5, 26-28, or 30; a polynucleotide comprising the full length protein coding sequence of SEQ ID NO: 1-3, 5, 26-28, or 30 (for example, SEQ ID NO: 4 or 29); and a polynucleotide comprising the nucleotide sequence of the mature protein coding sequence of any of SEQ ID NO: 1-3, 5, 26-28, or 30. The polynucleotides of the present invention also include, but are not limited to, a polynucleotide that hybridizes under stringent hybridization conditions to (a) the complement of any of the nucleotide sequences set forth in SEQ ID NO: 1-3, 5, 26-28, or 30; (b) a nucleotide sequence encoding any of SEQ ID NO: 4, 6-22, 25, 29, or 31-40; a polynucleotide which is an allelic variant of any polynucleotides recited above having at least 70% polynucleotide sequence identity to the polynucleotides; a polynucleotide which encodes a species homolog (e.g. orthologs) of any of the peptides recited above; or a polynucleotide that encodes a polypeptide comprising a specific domain or truncation of the polypeptide comprising SEQ ID NO: 4 or 29.
A collection as used in this application can be a collection of only one polynucleotide. The collection of sequence information or unique identifying information of each sequence can be provided on a nucleic acid array. In one embodiment, segments of sequence information are provided on a nucleic acid array to detect the polynucleotide that contains the segment. The array can be designed to detect full-match or mismatch to the polynucleotide that contains the segment. The collection can also be provided in a computer-readable format.
This invention further provides cloning or expression vectors comprising at least a fragment of the polynucleotides set forth above and host cells or organisms transformed with these expression vectors. Useful vectors include plasmids, cosmids, lambda phage derivatives, phagemids, and the like, that are well known in the art. Accordingly, the invention also provides a vector including a polynucleotide of the invention and a host cell containing the polynucleotide. In general, the vector contains an origin of replication functional in at least one organism, convenient restriction endonuclease sites, and a selectable marker for the host cell. Vectors according to the invention include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. A host cell according to the invention can be a prokaryotic or eukaryotic cell and can be a unicellular organism or part of a multicellular organism.
The compositions of the present invention include polypeptides comprising, but not limited to, an isolated polypeptide selected from the group comprising the amino acid sequence of SEQ ID NO: 4, 6-22, 25, 29, or 31-40; or the corresponding full length or mature protein. Polypeptides of the invention also include polypeptides with biological activity that are encoded by (a) any of the polynucleotides having a nucleotide sequence set forth in SEQ ID NO: 1-3, 5, 26-28, or 30; or (b) polynucleotides that hybridize to the complement of the polynucleotides of (a) under stringent hybridization conditions. Biologically or immunologically active variants of any of the protein sequences listed as SEQ ID NO: 4, 6-22, 25, 29, or 31-40 and substantial equivalents thereof that retain biological or immunological activity are also contemplated. The polypeptides of the invention may be wholly or partially chemically synthesized but are preferably produced by recombinant means using the genetically engineered cells (e.g. host cells) of the invention.
The invention also provides compositions comprising a polypeptide of the invention. Pharmaceutical compositions of the invention may comprise a polypeptide of the invention and an acceptable carrier, such as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
The invention also relates to methods for producing a polypeptide of the invention comprising culturing host cells comprising an expression vector containing at least a fragment of a polynucleotide encoding the polypeptide of the invention in a suitable culture medium under conditions permitting expression of the desired polypeptide, and purifying the protein or peptide from the culture or from the host cells. Preferred embodiments include those in which the protein produced by such a process is a mature form of the protein.
Polynucleotides according to the invention have numerous applications in a variety of techniques known to those skilled in the art of molecular biology. These techniques include use as hybridization probes, use as oligomers, or primers, for PCR, use in an array, use in computer-readable media, use for chromosome and gene mapping, use in the recombinant production of protein, and use in generation of antisense DNA or RNA, their chemical analogs and the like. For example, when the expression of an mRNA is largely restricted to a particular cell or tissue type, polynucleotides of the invention can be used as hybridization probes to detect the presence of the particular cell or tissue mRNA in a sample using, e.g., in situ hybridization.
In other exemplary embodiments, the polynucleotides are used in diagnostics as expressed sequence tags for identifying expressed genes or, as well known in the art and exemplified by Vollrath et al., Science 258:52-59 (1992), as expressed sequence tags for physical mapping of the human genome.
The polypeptides according to the invention can be used in a variety of conventional procedures and methods that are currently applied to other proteins. For example, a polypeptide of the invention can be used to generate an antibody that specifically binds the polypeptide. Such antibodies, particularly monoclonal antibodies, are useful for detecting or quantitating the polypeptide in tissue. The polypeptides of the invention can also be used as molecular weight markers, and as a food supplement.
Methods are also provided for preventing, treating, or ameliorating a medical condition which comprises the step of administering to a mammalian subject a therapeutically effective amount of a composition comprising a peptide of the present invention and a pharmaceutically acceptable carrier.
Thus, the alpha-2-macroglobulin-like polypeptides and polynucleotides of the invention may be used in the treatment of Alzheimer""s disease and cerebral amyloid angiopathy, multiple sclerosis, congenital antithrombin deficiency, rheumatoid arthritis and growth of various tumors, coronary or limb ischemia, retinopathies, and regulation of immune response to tumors and viral infections.
The methods of the invention also provide methods for the treatment of disorders as recited herein which comprise the administration of a therapeutically effective amount of a composition comprising a polynucleotide or polypeptide of the invention and a pharmaceutically acceptable carrier to a mammalian subject exhibiting symptoms or tendencies related to disorders as recited herein. In addition, the invention encompasses methods for treating diseases or disorders as recited herein comprising the step of administering a composition comprising compounds and other substances that modulate the overall activity of the target gene products and a pharmaceutically acceptable carrier. Compounds and other substances can effect such modulation either on the level of target gene/protein expression or target protein activity. Specifically, methods are provided for preventing, treating or ameliorating a medical condition, including viral diseases, which comprises administering to a mammalian subject, including but not limited to humans, a therapeutically effective amount of a composition comprising a polypeptide of the invention or a therapeutically effective amount of a composition comprising a binding partner of (e.g., antibody specifically reactive for) alpha-2-macroglobulin-like polypeptides of the invention. The mechanics of the particular condition or pathology will dictate whether the polypeptides of the invention or binding partners (or inhibitors) of these would be beneficial to the individual in need of treatment.
According to this method, polypeptides of the invention can be administered to produce an in vitro or in vivo inhibition of cellular function. A polypeptide of the invention can be administered in vivo alone or as an adjunct to other therapies. Conversely, protein or other active ingredients of the present invention may be included in formulations of a particular agent to minimize side effects of such an agent.
The invention further provides methods for manufacturing medicaments useful in the above-described methods.
The present invention further relates to methods for detecting the presence of the polynucleotides or polypeptides of the invention in a sample (e.g., tissue or sample). Such methods can, for example, be utilized as part of prognostic and diagnostic evaluation of disorders as recited herein and for the identification of subjects exhibiting a predisposition to such conditions.
The invention provides a method for detecting a polypeptide of the invention in a sample comprising contacting the sample with a compound that binds to and forms a complex with the polypeptide under conditions and for a period sufficient to form the complex and detecting formation of the complex, so that if a complex is formed, the polypeptide is detected.
The invention also provides kits comprising polynucleotide probes and/or monoclonal antibodies, and optionally quantitative standards, for carrying out methods of the invention. Furthermore, the invention provides methods for evaluating the efficacy of drugs, and monitoring the progress of patients, involved in clinical trials for the treatment of disorders as recited above.
The invention also provides methods for the identification of compounds that modulate (i.e., increase or decrease) the expression or activity of the polynucleotides and/or polypeptides of the invention. Such methods can be utilized, for example, for the identification of compounds that can ameliorate symptoms of disorders as recited herein. Such methods can include, but are not limited to, assays for identifying compounds and other substances that interact with (e.g., bind to) the polypeptides of the invention.
The invention provides a method for identifying a compound that binds to the polypeptide of the present invention comprising contacting the compound with the polypeptide under conditions and for a time sufficient to form a polypeptide/compound complex and detecting the complex, so that if the polypeptide/compound complex is detected, a compound that binds to the polypeptide is identified.
Also provided is a method for identifying a compound that binds to the polypeptide comprising contacting the compound with the polypeptide in a cell for a time sufficient to form a polypeptide/compound complex wherein the complex drives expression of a reporter gene sequence in the cell and detecting the complex by detecting reporter gene sequence expression so that if the polypeptide/compound complex is detected a compound that binds to the polypeptide is identified.